PROJECT SUMMARY Sophisticated, facile, and potent inhibitors of Cas enzymes that are simple to use are still a major missing tool for CRISPR-based biomedical and therapeutic research. The rationale for CRISPR-Cas inhibitors includes a need for more efficient generation of model cells and organisms, the threat of dangerous off-target editing in CRISPR-based therapeutics, and the desire for more precise control over Cas enzyme activity in synthetic biology and diagnostics applications. Methods and modified Cas enzymes have been developed to help address these issues and other shortcomings of CRISPR-Cas systems, particularly Cas9 from Streptococcus pyogenes (SpCas9). These include Cas9 mutants with lower off-target activity and Cas9 mutants and protein fusions that allow small molecule or light-based activation of Cas9. However, these modifications typically have a concomitant decrease in on-target activity and do not address the need for rapid and specific shut-down of Cas9. Despite improvements to Cas enzymes, the need for specific, broadly applicable, and easy-to-use inhibitors will remain a necessary tool that is not available. Small molecules that can inhibit Cas9 may take significant effort to develop and potentially come with downsides, including their own off-target effects and inadvertent impacts on the organism?s microbiota, possibly similar to antibiotic side-effects. Natural anti-CRISPR proteins have recently been discovered that bind Cas9 and other Cas proteins with high affinity. While promising as inhibitors, they are too large to possess drug-like properties and their minimization or optimization is not an obvious exercise. They must be genetically encoded for use in cells or organisms. However, they provide inspiration for the design of a new class of CRISPR-Cas inhibitors. Here we propose to develop a new technology, nucleic acid-based (NAB) inhibitors of Cas9 enzymes. These molecules are smaller than natural anti-CRISPR proteins but can bind with similar affinity, be chemically synthesized, and be readily introduced into cells with common methods. In this project, we will develop NAB inhibitor technology by optimizing their size, chemistry, binding affinity, inhibitory activity, and cellular stability. We will further develop methods and molecules that facilitate direct, carrier-free delivery and timed-release of NAB inhibitors. The resulting NAB inhibitors are expected to be broadly applicable and straightforward to use for diverse biomedical research. We expect NAB inhibitors to become valuable fail-safe inhibitors to overcome the critical safety hurdles in CRISPR-based therapeutics.